Pertussis or whooping cough is a life threatening infectious disease that is now spreading in the highly vaccinated populations of the most developed countries. This is largely due to use of safe but less efficient acellular pertussis vaccines that were developed without the understanding of mechanisms by which Bordetella pertussis suppresses and evades host immunity. We shall test herein the hypothesis that adenylate cyclase toxin (ACT) action suppresses adaptive cellular immune responses on respiratory mucosa through subversion of maturation of intraepithelial dendritic cells (DCs). Phenotype of DCs migrating from infected mucosa into draining lymph nodes shall be examined and the capacity of migrating DCs to activate antigen-specific T cells and/or suppress immune responses and expand of tolerogenic Treg cells will be determined. The acquired knowledge will be used to develop a next generation of immunogenic and safe pertussis vaccines consisting of suspensions of genetically detoxified bacteria, admixed with toxoids of ACT and PT.

Černý kašel (pertuse) je život ohrožující infekční onemocnění, které se opět šíří ve vysoce proočkovaných populacích nejvyspělejších zemí, jež používají bezpečné, ale málo účinné acelulární pertusové vakcíny. Tyto byly vyvinuty bez dostatečné znalosti mechanismů, jimiž bakterie Bordetella pertussis potlačuje vrozenou a adaptivní imunitu hostitele. V tomto projektu otestujeme hypotézu, že působení adenylát-cyklázového toxinu (ACT) potlačuje adaptivní buněčnou imunitní odpověď hostitele na infekci. Pokročilými zobrazovacími metodami, a s využitím specifických mutant, prověříme působení ACT na maturaci intraepitheliálních dendritických buněk. Prostudujeme fenotyp dendritických buněk migrujících z infikované sliznice do mízních uzlin a stanovíme jejich schopnost aktivovat T lymfocyty a/nebo expandovat regulační T buňky a tlumit imunitní odpověď. Toto poznání uplatníme při vývoji vysoce imunogenních a bezpečných experimentálních pertusových vakcín nové generace, založených na vícečetně geneticky detoxifikovaných bakteriálních buňkách ve směsi s toxoidy ACT a PT.

• MeSH
• adenylátcyklasový toxin imunologie   škodlivé účinky   MeSH
• dendritické buňky imunologie   MeSH
• fenotyp MeSH
• imunosupresiva škodlivé účinky   MeSH
• lidé MeSH
• pertuse imunologie   prevence a kontrola   MeSH
• pertusová vakcína MeSH
• T-lymfocyty MeSH
• testované léky MeSH
• vyvíjení léků MeSH
• Check Tag
• lidé MeSH

• Konspekt
• Patologie. Klinická medicína
• NLK Obory
• alergologie a imunologie
• pneumologie a ftizeologie
• preventivní medicína
• NLK Publikační typ
• závěrečné zprávy o řešení grantu AZV MZ ČR

Monocytes arriving at the site of infection differentiate into functional effector macrophages to replenish the resident sentinel cells. Bordetella pertussis, the pertussis agent, secretes an adenylate cyclase toxin-hemolysin (CyaA) that binds myeloid phagocytes through complement receptor 3 (CD11b/CD18) and swiftly delivers its adenylyl cyclase enzyme domain into phagocytes. This ablates the bactericidal capacities of phagocytes through massive and unregulated conversion of cytosolic ATP into the key signaling molecule cAMP. We show that exposure of primary human monocytes to as low a concentration as 22.5 pM CyaA, or a low (2:1) multiplicity of infection by CyaA-producing B. pertussis bacteria, blocks macrophage colony-stimulating factor (M-CSF)-driven differentiation of monocytes. CyaA-induced cAMP signaling mediated through the activity of protein kinase A (PKA) efficiently blocked expression of macrophage markers, and the monocytes exposed to 22.5 pM CyaA failed to acquire the characteristic intracellular complexity of mature macrophage cells. Neither M-CSF-induced endoplasmic reticulum (ER) expansion nor accumulation of Golgi bodies, mitochondria, or lysosomes was observed in toxin-exposed monocytes, which remained small and poorly phagocytic and lacked pseudopodia. Exposure to 22.5 pM CyaA toxin provoked loss of macrophage marker expression on in vitro differentiated macrophages, as well as on primary human alveolar macrophages, which appeared to dedifferentiate into monocyte-like cells with upregulated CD14 levels. This is the first report that terminally differentiated tissue-resident macrophage cells can be dedifferentiated in vitro The results suggest that blocking of monocyte-to-macrophage transition and/or dedifferentiation of the sentinel cells of innate immunity through cAMP-elevating toxin action may represent a novel immune evasion strategy of bacterial pathogens.IMPORTANCE Macrophages are key sentinel cells of the immune system, and, as such, they are targeted by the toxins produced by the pertussis agent Bordetella pertussis The adenylate cyclase toxin (CyaA) mediates immune evasion of B. pertussis by suspending the bactericidal activities of myeloid phagocytes. We reveal a novel mechanism of potential subversion of host immunity, where CyaA at very low (22 pM) concentrations could inhibit maturation of human monocyte precursors into the more phagocytic macrophage cells. Furthermore, exposure to low CyaA amounts has been shown to trigger dedifferentiation of mature primary human alveolar macrophages back into monocyte-like cells. This unprecedented capacity is likely to promote survival of the pathogen in the airways, both by preventing maturation of monocytes attracted to the site of infection into phagocytic macrophages and by dedifferentiation of the already airway-resident sentinel cells.

• MeSH
• adenylátcyklasový toxin škodlivé účinky   metabolismus   MeSH
• alveolární makrofágy účinky léků   metabolismus   MeSH
• Bordetella pertussis chemie   MeSH
• buněčná diferenciace účinky léků   MeSH
• interakce hostitele a patogenu MeSH
• lidé MeSH
• monocyty účinky léků   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The adenylate cyclase toxin-hemolysin (CyaA, ACT, or AC-Hly) plays a crucial role in virulence and airway colonization capacity of the whooping cough agent Bordetella pertussis. The toxin penetrates target cell membranes and exhibits three distinct biological activities. A population of CyaA conformers forms small cation-selective pores that permeabilize the cell membrane for potassium efflux, which can provoke colloid-osmotic (oncotic) cell lysis. The other two activities are due to CyaA conformers that transiently form calcium influx conduits in the target cell membrane and translocate the adenylate cyclase (AC) enzyme into cytosol of cells. A fourth putative biological activity has recently been reported; an intrinsic phospholipase A (PLA) activity was claimed to be associated with the CyaA polypeptide and be involved in the mechanism of translocation of the AC enzyme polypeptide across cell membrane lipid bilayer. However, the conclusions drawn by the authors contradicted their own results and we show them to be erroneous. We demonstrate that highly purified CyaA is devoid of any detectable phospholipase A1 activity and that contrary to the published claims, the two putative conserved phospholipase A catalytic residues, namely the Ser606 and Asp1079 residues, are not involved in the process of membrane translocation of the AC domain of CyaA across target membranes.

• MeSH
• adenylátcyklasový toxin metabolismus   toxicita   MeSH
• Bordetella pertussis MeSH
• buněčné linie MeSH
• erytrocyty MeSH
• fosfolipasy A metabolismus   MeSH
• hemolýza MeSH
• kyselina asparagová MeSH
• myši MeSH
• ovce MeSH
• serin MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The airway epithelium restricts the penetration of inhaled pathogens into the underlying tissue and plays a crucial role in the innate immune defense against respiratory infections. The whooping cough agent, Bordetella pertussis, adheres to ciliated cells of the human airway epithelium and subverts its defense functions through the action of secreted toxins and other virulence factors. We examined the impact of B. pertussis infection and of adenylate cyclase toxin-hemolysin (CyaA) action on the functional integrity of human bronchial epithelial cells cultured at the air-liquid interface (ALI). B. pertussis adhesion to the apical surface of polarized pseudostratified VA10 cell layers provoked a disruption of tight junctions and caused a drop in transepithelial electrical resistance (TEER). The reduction of TEER depended on the capacity of the secreted CyaA toxin to elicit cAMP signaling in epithelial cells through its adenylyl cyclase enzyme activity. Both purified CyaA and cAMP-signaling drugs triggered a decrease in the TEER of VA10 cell layers. Toxin-produced cAMP signaling caused actin cytoskeleton rearrangement and induced mucin 5AC production and interleukin-6 (IL-6) secretion, while it inhibited the IL-17A-induced secretion of the IL-8 chemokine and of the antimicrobial peptide beta-defensin 2. These results indicate that CyaA toxin activity compromises the barrier and innate immune functions of Bordetella-infected airway epithelia.

• MeSH
• adenylátcyklasový toxin genetika   metabolismus   toxicita   MeSH
• AMP cyklický metabolismus   MeSH
• Bordetella pertussis genetika   metabolismus   MeSH
• bronchy cytologie   metabolismus   mikrobiologie   MeSH
• cytoskelet metabolismus   MeSH
• epitelové buňky metabolismus   mikrobiologie   MeSH
• interleukin-6 metabolismus   MeSH
• lidé MeSH
• mucin 5AC metabolismus   MeSH
• pertuse genetika   metabolismus   mikrobiologie   MeSH
• signální transdukce účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The adenylate cyclase toxin (CyaA) of the whooping cough agent Bordetella pertussis subverts immune functions of host myeloid cells expressing the αMβ2 integrin (CD11b/CD18, CR3 or Mac-1). CyaA delivers into cytosol of cells an extremely catalytically active adenylyl cyclase enzyme, which disrupts the innate and adaptive immune functions of phagocytes through unregulated production of the key signaling molecule cAMP. We have used phosphoproteomics to analyze cAMP signaling of CyaA in murine bone marrow-derived dendritic cells. CyaA action resulted in alterations of phosphorylation state of a number of proteins that regulate actin cytoskeleton homeostasis, including Mena, Talin-1 and VASP. CyaA action repressed mTOR signaling through activation of mTORC1 inhibitors TSC2 and PRAS40 and altered phosphorylation of multiple chromatin remodelers, including the class II histone deacetylase HDAC5. CyaA toxin action further elicited inhibitory phosphorylation of SIK family kinases involved in modulation of immune response and provoked dephosphorylation of the transcriptional coactivator CRTC3, indicating that CyaA-promoted nuclear translocation of CRTC3 may account for CyaA-induced IL-10 production. These findings document the complexity of subversive physiological manipulation of myeloid phagocytes by the CyaA toxin, serving in immune evasion of the pertussis agent.

• MeSH
• AMP cyklický metabolismus   MeSH
• Bordetella pertussis metabolismus   MeSH
• cytoskeletální proteiny metabolismus   MeSH
• dendritické buňky metabolismus   MeSH
• fosfoproteiny metabolismus   MeSH
• histondeacetylasy metabolismus   MeSH
• mikrofilamentové proteiny metabolismus   MeSH
• molekuly buněčné adheze metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• pertuse mikrobiologie   MeSH
• signální transdukce fyziologie   MeSH
• talin metabolismus   MeSH
• transkripční faktory metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Bordetellae, pathogenic to mammals, produce an immunomodulatory adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) that enables them to overcome the innate immune defense of the host. CyaA subverts host phagocytic cells by an orchestrated action of its functional domains, where an extremely catalytically active adenylyl cyclase enzyme is delivered into phagocyte cytosol by a pore-forming repeat-in-toxin (RTX) cytolysin moiety. By targeting sentinel cells expressing the complement receptor 3, known as the CD11b/CD18 (αMβ₂) integrin, CyaA compromises the bactericidal functions of host phagocytes and supports infection of host airways by Bordetellae. Here, we review the state of knowledge on structural and functional aspects of CyaA toxin action, placing particular emphasis on signaling mechanisms by which the toxin-produced 3',5'-cyclic adenosine monophosphate (cAMP) subverts the physiology of phagocytic cells.

• MeSH
• adenylátcyklasový toxin chemie   MeSH
• alveolární makrofágy cytologie   MeSH
• AMP cyklický chemie   MeSH
• Bordetella pertussis MeSH
• dendritické buňky cytologie   MeSH
• fagocyty chemie   MeSH
• kinasa Syk MeSH
• lidé MeSH
• makrofágový antigen 1 MeSH
• neutrofily cytologie   MeSH
• proteinové domény MeSH
• signální transdukce * MeSH
• terciární struktura proteinů MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

Adenylate cyclase toxin (CyaA) is released in the course of B. pertussis infection in the host's respiratory tract in order to suppress its early innate and subsequent adaptive immune defense. CD11b-expressing dendritic cells (DC), macrophages and neutrophils are professional phagocytes and key players of the innate immune system that provide a first line of defense against invading pathogens. Recent findings revealed the capacity of B. pertussis CyaA to intoxicate DC with high concentrations of 3',5'-cyclic adenosine monophosphate (cAMP), which ultimately skews the host immune response towards the expansion of Th17 cells and regulatory T cells. CyaA-induced cAMP signaling swiftly incapacitates opsonophagocytosis, oxidative burst and NO-mediated killing of bacteria by neutrophils and macrophages. The subversion of host immune responses by CyaA after delivery into DC, macrophages and neutrophils is the subject of this review.

• MeSH
• adenylátcyklasový toxin imunologie   MeSH
• AMP cyklický chemie   MeSH
• Bordetella pertussis MeSH
• buněčná imunita MeSH
• dendritické buňky imunologie   MeSH
• dýchací soustava imunologie   mikrobiologie   MeSH
• fagocytóza MeSH
• interakce hostitele a patogenu MeSH
• lidé MeSH
• makrofágy imunologie   MeSH
• neutrofily imunologie   MeSH
• pertuse imunologie   MeSH
• regulační T-lymfocyty imunologie   MeSH
• signální transdukce MeSH
• slizniční imunita MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

The adenylate cyclase toxin-hemolysin (CyaA, ACT, or AC-Hly) of Bordetella pertussis targets phagocytic cells expressing the complement receptor 3 (CR3, Mac-1, αMβ2 integrin, or CD11b/CD18). CyaA delivers into cells an N-terminal adenylyl cyclase (AC) enzyme domain that is activated by cytosolic calmodulin and catalyzes unregulated conversion of cellular ATP into cyclic AMP (cAMP), a key second messenger subverting bactericidal activities of phagocytes. In parallel, the hemolysin (Hly) moiety of CyaA forms cation-selective hemolytic pores that permeabilize target cell membranes. We constructed the first B. pertussis mutant secreting a CyaA toxin having an intact capacity to deliver the AC enzyme into CD11b-expressing (CD11b(+)) host phagocytes but impaired in formation of cell-permeabilizing pores and defective in cAMP elevation in CD11b(-) cells. The nonhemolytic AC(+) Hly(-) bacteria inhibited the antigen-presenting capacities of coincubated mouse dendritic cells in vitro and skewed their Toll-like receptor (TLR)-triggered maturation toward a tolerogenic phenotype. The AC(+) Hly(-) mutant also infected mouse lungs as efficiently as the parental AC(+) Hly(+) strain. Hence, elevation of cAMP in CD11b(-) cells and/or the pore-forming capacity of CyaA were not required for infection of mouse airways. The latter activities were, however, involved in bacterial penetration across the epithelial layer, enhanced neutrophil influx into lung parenchyma during sublethal infections, and the exacerbated lung pathology and lethality of B. pertussis infections at higher inoculation doses (>10(7) CFU/mouse). The pore-forming activity of CyaA further synergized with the cAMP-elevating activity in downregulation of major histocompatibility complex class II (MHC-II) molecules on infiltrating myeloid cells, likely contributing to immune subversion of host defenses by the whooping cough agent.

• MeSH
• adenylátcyklasový toxin metabolismus   MeSH
• AMP cyklický metabolismus   MeSH
• antigeny CD11b metabolismus   MeSH
• Bordetella pertussis patogenita   MeSH
• buněčná membrána metabolismus   MeSH
• dendritické buňky imunologie   MeSH
• fagocyty imunologie   MeSH
• hemolyziny metabolismus   MeSH
• makrofágový antigen 1 metabolismus   MeSH
• myši inbrední BALB C MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• pertuse mikrobiologie   MeSH
• plíce mikrobiologie   patologie   MeSH
• T-lymfocyty imunologie   MeSH
• virulence MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH